Many attempts to drastically modify bacteria for use in industry have failed since such modifications are most of the time lethal or lead to uncompetitive and unstable organisms.
For example; in eubacteria, peptide synthesis is initiated at methionine start codons which are read by N-formyl methionine TRNA. Prior to translation initiation the methionyl moiety of the charged tRNA is N-formylated by the action of Met-tRNAi transformylase. The N-formyl group is removed from the native protein by polypeptide deformylase (E.C. 3.5.1.27), and the initiator methionine can then be cleaved off by methionine aminopeptidase, completing the primer methionine cycle. In contrast, archaea and eukaryotes have a primer methionine cycle devoid of N-formylating and deformylating activities (for review see Mazel et al., 1994, 1996).
Expression of eukaryotic proteins in eubacterial hosts often results in the production of recombinant proteins that retain an N-terminal formylmethionyl residue (examples include bovine somatotropin [Bogosian et al., 1989]; eel growth hormone [Sugimoto et al., 1990]; human granulocyte colony-stimulating factor [Clogston et al., 1992]; bovine fatty acid-binding protein [Specht, et al., 1994]; bovine cytochrome P450 [Dong et al. 1995]; Methanothermus fervidus histone A [Sandman et al., 1995]; human interleukin-5 [Rose et al., 1992]; human parathyroid hormone [Rabbani et al., 1988; Hogset et al., 1990]; human gamma-interferon [Honda et al., 1989]). In addition retention of N-formyl methionine has been found in endogeneous E. coli proteins (Hauschild-Rogat, 1968; Marasco et al., 1984; Milligan and Koshland, 1990).
Since N-formylated peptides are a major indicator of eubacterial infections for the mammalian immune system and are highly immunogenic, incomplete deformylation precludes, for example, the use of N-formylated preparations for therapeutic purposes.
Several approaches to circumvent this problem have been proposed, e.g., expression in the presence of trimethoprim and thymidine (Sandman et al., 1995), overexpression of peptide deformylase in the host (U.S. Pat. No. 6,190,902), expression as a protein fusion either with an N-terminal peptide that can be removed in vitro by a specific protease or with an N-terminal leader peptide which is cleaved during transport of the nascent protein in a non-cytoplasmatic compartment. Finally, the N-formyl group may also be removed by mild acid hydrolysis, or the fraction of the protein retaining N-formyl methionine may be separated from the correctly processed protein by purification procedures.
Each of these approaches has significant disadvantages. Addition of trimethoprim and thymidine is costly, requires manipulation of the culture that will express the recombinant protein, and may slow down growth of the host. Overexpression of peptide deformylase requires a stable plasmid construct. in the host that has to be selected for; moreover, deformylation may be less than 100% effective. Expression of fusion proteins requires exact molecular constructions; chemical hydrolysis with acid may cause damage to the rest of the protein. Finally, none of these approaches guarantees a final preparation that is absolutely free of N-formylated peptides derived either from the recombinant protein or from contaminations with endogeneous host peptides.
The above mentioned drawbacks are solved if one could produce peptides in bacteria that are freed of the N-transformylation system. But, for bacteria that have evolved with this system for billions of year, removal of this system would normally result in death or serious impairment ultimately leading to organisms that are uncompetitive and genetically unstable. Indeed, when these modified bacteria are in contact with non modified bacteria they either recuperate the lost functional genetic elements or they simply disappear in favor of the more competitive natural bacteria.
One could also think that it would take billions of years of evolution to see emergence of bacteria devoid of the N-transformylation activity.
In connection with the invention, the N-transformylation system have been inactivated in bacteria having normally the N-transformylation system. Following a selective process, strains that are genetically stable and capable of competing with the natural bacteria have been obtained in only one month.
The invention opens new possibilities for obtaining new organisms that will constitute new species useful in all kind of industries by means of resurgent evolution.